Invariant Natural Killer T (iNKT) cells are a distinct subset of T lymphocytes that are activated by TCR engagement of lipid, rather than peptide, antigens presented by the MHC Class I-like molecule CD1d. Endogenous glycolipids, most likely derived from oxidized lipoproteins, engulfed apoptotic cells, or induced by infection, are loaded onto CD1d to selectively activate iNKT cells. Upon activation by CD1d, iNKT cells release large quantities of IFN3, IL-4, and IL-13. This CD1d- iNKT cell axis constitutes a very rapid, innate immune response. ABCG1 is an ATP-binding cassette transporter that effluxes cholesterol from peripheral cells to HDL for reverse cholesterol transport. ABCG1 expression is ubiquitous, with high expression in dendritic cells (DC) and lymphocytes. Our preliminary data show that a) ABCG1-/- mice possess very few iNKT cells in thymus, supporting the concept of a defect in thymic development;b) the defect in thymic iNKT development is caused by cell-autonomous changes in iNKT precursors mediated by absence of ABCG1;c) antigen presenting cells from mice that have selective deletion of ABCG1 in CD11c+ cells show reduced surface expression of CD1d and have reduced ability to activate iNKT cells in vivo;and d) ABCG1 resides primarily intracellularly and functions to regulate endosomal/lysosomal membrane dynamics. Thus, we hypothesize that ABCG1 impacts NKT cell biology in both a NKT cell-autonomous manner and by contributing to functional changes in CD11c+ cells that modulate NKT activation. We will test these hypotheses in the following 3 specific aims. Specific Aim 1 will test the hypothesis that ABCG1 modulates iTCR signaling for proliferation or survival of iNKT thymic precursors. Specific Aim 2 will test the hypothesis that ABCG1 modulates CD1d function in antigen presenting cells, impacting iNKT activation. Aim 2 will also test the role of ABCG1 on iNKT cell-autonomous activation in the periphery. Aim 3 will test whether absence of ABCG1 in iNKT cells confers atheroprotection in mice by reducing iNKT numbers and activation in vivo. Regulation of iNKT cell production and activation by ABCG1 is a novel, unexplored role for ABCG1 in immunity that could have profound effects on diseases such as atherosclerosis, rheumatoid arthritis, allergy, and autoimmunity. PUBLIC HEALTH RELEVANCE: Invariant Natural Killer T (iNKT) cells are a unique subset of lymphocytes that are activated by lipid, rather than protein, antigens. Activation of iNKT cells results in rapid release of cytokines that regulate the immune response to infection. Although iNKT cells are important in combating infection, they also can unfortunately contribute to chronic diseases, such as autoimmunity, rheumatoid arthritis, and atherosclerosis, if the iNKT cells themselves become activated chronically. This project will test a novel role for a cholesterol transporter, ABCG1, in regulating the production and activation of iNKT cells. Mice deficient in ABCG1 have reduced numbers and chronic activation of iNKT cells and are protected from atherosclerosis. The goals of the project are to determine mechanisms for how ABCG1 impacts iNKT production and activation, and to test if decreased iNKT cell activation by ABCG1 deficiency results in atheroprotection in vivo.